Best practices for user consultation in flow cytometry shared resource laboratories.

This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation.

Reporting and conducting patient journey mapping research in healthcare: A scoping review.

AIM: To identify how patient journey mapping is being undertaken and reported. DESIGN: A scoping review of the literature was undertaken using JBI guidance. DATA SOURCES: Databases were searched in July 2021 (16th-21st), including Ovid's Medline, Embase, Emcare and PsycINFO; Scopus; Web of Science Core Collection, the Directory of Open Access Journals; Informit and; ProQuest Dissertations and Theses Global. REVIEW METHODS: Eligible articles included peer-reviewed literature documenting journey mapping methodologies and studies conducted in healthcare services. Reviewers used Covidence to screen titles and abstracts of located sources, and to screen full-text articles. A table was used to extract data and synthesize results. RESULTS: Eighty-one articles were included. An acceleration of patient journey mapping research was observed, with 76.5% (n = 62) of articles published since 2015. Diverse mapping approaches were identified. Reporting of studies was inconsistent and largely non-adherent with relevant, established reporting guidelines. CONCLUSION: Patient journey mapping is a relatively novel approach for understanding patient experiences and is increasingly being adopted. There is variation in process details reported. Considerations for improving reporting standards are provided. IMPACT: Patient journey mapping is a rapidly growing approach for better understanding how people enter, experience and exit health services. This type of methodology has significant potential to inform new, patient centred models of care and facilitate clinicians, patients and health professionals to better understand gaps and strategies in health services. The synthesised results of this review alert researchers to options available for journey mapping research and provide preliminary guidance for elevating reporting quality.

Considerations for user consultation in a flow cytometry shared resource laboratory.

User consultation is an essential first step in assuring high-quality flow cytometric data. A central challenge to shared resource laboratory (SRL) staff is how to best guide new and current users to meet each projects' needs. One solution to this challenge is to follow a standard user consultation platform addressing all critical steps between the conception of the experiment and the actual acquisition of samples. Here we describe considerations to help an SRL understand the researcher's goals and how best the SRL staff can provide expert advice in a structured manner. User consultation has an educational nature, informing users about current best practices in cytometry that apply to their specific utilization. A consultation report also improves communication between the SRL, principal investigator, and lab members of the collaborating researcher. Development of best SRL practices is spearheaded by the ISAC SRL committee and this communication sets the foundation to initiate such report for user consultation. Implementation of best practices during user consultation will improve rigor and reproducibility in cytometry.

Using Simulation to Assess Nurse Practitioner Education.

ABSTRACT: Resource restraints and the movement toward competency-based education encourage exploration of innovative simulation experiences in advanced practice registered nurse education. Building upon existing best practices in simulation development, this pilot project explores opportunities and challenges translating these practices to the advanced practice registered nurse context. Innovations focused on using a competency-based framework, developing a competency-based evaluation tool, and providing feedback from faculty and standardized patients. Lessons learned from this experience inform recommendations for how to design instructional experiences and provide formative methods of feedback for nurse practitioner student assessment.

Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria.

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations. We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment. We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis. In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

Efficacy of mindfulness-based stress reduction in emerging adults with poorly controlled, type 1 diabetes: A pilot randomized controlled trial.

BACKGROUND/OBJECTIVE: The negative effects of stress on persons with type 1 diabetes (T1D) are well-established, but effective interventions to reduce stress among emerging adults with T1D are limited. The study objective was to conduct a pilot randomized controlled trial (RCT) to obtain preliminary data on the efficacy of mindfulness-based stress reduction (MBSR) to reduce stress and improve diabetes health outcomes in a population of high-risk, urban emerging adults with poorly controlled diabetes. METHODS: Forty-eight participants aged 16 to 20 years of age with T1D (mean duration = 8 years) were randomly assigned to one of three conditions: MSBR, cognitive-behavioral stress management (CBSM), or a diabetes support group. Data were collected at baseline, end of treatment, and 3 months after treatment completion. Measures of self-reported stress and depressive symptoms, diabetes management, and glycemic control were obtained. RESULTS: MBSR was found to reduce self-reported stress at end of treatment (P = 0.03, d = -0.49) and 3-month follow-up (P = 0.01, d = -0.67), but no effects on diabetes management or glycemic control were found. Diabetes support group participants had improved glycemic control at the end of treatment (P = 0.01, d = -0.62) as well as reduced depressive symptoms at 3-month follow-up (P = 0.01, d = -0.71). CONCLUSIONS: Results provide preliminary support for the efficacy of MBSR to improve psychosocial adjustment in emerging adults with poorly controlled T1D but require replication in adequately powered studies. Findings also support the value of peer support in improving health outcomes in this age group.

Cutting Edge: IL-10 Is Essential for the Generation of Germinal Center B Cell Responses and Anti-Plasmodium Humoral Immunity.

IL-10 is a pleiotropic cytokine expressed during malaria, a disease characterized by short-lived, parasite-specific Ab responses. The role of IL-10 in regulating B cell responses during malaria is not known. In this study we report that IL-10 is essential for anti-Plasmodium humoral immunity. We identify that germinal center (GC) B cell reactions, isotype-switched Ab responses, parasite control, and host survival require B cell-intrinsic IL-10 signaling. IL-10 also indirectly supports humoral immunity by suppressing excessive IFN-γ, which induces T-bet expression in B cells. Genetic ablation of either IFN-γ signaling or T-bet expression in B cells substantially enhanced GC B cell responses and anti-Plasmodium Ab production. Together, our data show that B cell-intrinsic IL-10 enhances whereas B cell-intrinsic IFN-γ and T-bet suppress GC B cell responses and anti-Plasmodium humoral immunity. These data identify critical immunoregulatory circuits in B cells that may be targeted to promote long-lived humoral immunity and resistance to malaria.

Type I Interferons Induce T Regulatory 1 Responses and Restrict Humoral Immunity during Experimental Malaria.

CD4 T cell-dependent antibody responses are essential for limiting Plasmodium parasite replication and the severity of malaria; however, the factors that regulate humoral immunity during highly inflammatory, Th1-biased systemic infections are poorly understood. Using genetic and biochemical approaches, we show that Plasmodium infection-induced type I interferons limit T follicular helper accumulation and constrain anti-malarial humoral immunity. Mechanistically we show that CD4 T cell-intrinsic type I interferon signaling induces T-bet and Blimp-1 expression, thereby promoting T regulatory 1 responses. We further show that the secreted effector cytokines of T regulatory 1 cells, IL-10 and IFN-γ, collaborate to restrict T follicular helper accumulation, limit parasite-specific antibody responses, and diminish parasite control. This circuit of interferon-mediated Blimp-1 induction is also operational during chronic virus infection and can occur independently of IL-2 signaling. Thus, type I interferon-mediated induction of Blimp-1 and subsequent expansion of T regulatory 1 cells represent generalizable features of systemic, inflammatory Th1-biased viral and parasitic infections that are associated with suppression of humoral immunity.

Commercial Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) Testing is Not Vulnerable to Incidental Alcohol Exposure in Pregnant Women.

BACKGROUND: Ethyl Glucoronide (EtG) and Ethyl Sulfate (EtS) have shown promise as biomarkers for alcohol and may be sensitive enough for use with pregnant women in whom even low-level alcohol use is important. However, there have been reports of over-sensitivity of EtG and EtS to incidental exposure to sources such as alcohol-based hand sanitizer. Further, few studies have evaluated these biomarkers among pregnant women, in whom the dynamics of these metabolites may differ. OBJECTIVES: This study evaluated whether commercial EtG-EtS testing was vulnerable to high levels of environmental exposure to alcohol in pregnant women. METHODS: Two separate samples of five nurses-one pregnant and the other postpartum, all of whom reported high levels of alcohol-based hand sanitizer use-provided urine samples before and 4-8 hours after rinsing with alcohol-based mouthwash and using hand sanitizer. The five pregnant nurses provided urine samples before, during, and after an 8-hour nursing shift, during which they repeatedly cleansed with alcohol-based hand sanitizer (mean 33.8 uses). The five postpartum nurses used hand sanitizer repeatedly between baseline and follow-up urine samples. RESULTS: No urine samples were positive for EtG-EtS at baseline or follow-up, despite use of mouthwash and-in the pregnant sample-heavy use of hand sanitizer (mean of 33.8 uses) throughout the 8-hour shift. CONCLUSIONS/IMPORTANCE: Current, commercially available EtG-EtS testing does not appear vulnerable to even heavy exposure to incidental sources of alcohol among pregnant and postpartum women.

Inflammatory response of mast cells during influenza A virus infection is mediated by active infection and RIG-I signaling.

Influenza A virus (IAV) is a major respiratory pathogen of both humans and animals. The lung is protected from pathogens by alveolar epithelial cells, tissue-resident alveolar macrophages, dendritic cells, and mast cells. The role of alveolar epithelial cells, endothelial cells, and alveolar macrophages during IAV infection has been studied previously. In this study, we address the role of mast cells during IAV infection. Respiratory infection with A/WSN/33 causes significant disease and immunopathology in C57BL/6 mice but not in B6.Cg-Kit(W-sh) mice, which lack mast cells. During in vitro coculture, A/WSN/33 caused mast cells to release histamine, secrete cytokines and chemokines, and produce leukotrienes. Moreover, when mast cells were infected with IAV, the virus did not replicate within mast cells. Importantly, human H1N1, H3N2, and influenza B virus isolates also could activate mast cells in vitro. Mast cell production of cytokines and chemokines occurs in a RIG-I/MAVS-dependent mechanism; in contrast, histamine production occurred through a RIG-I/MAVS-independent mechanism. Our data highlight that, following IAV infection, the response of mast cells is controlled by multiple receptors. In conclusion, we identified a unique inflammatory cascade activated during IAV infection that could potentially be targeted to limit morbidity following IAV infection.

Using short-read 16S rRNA sequencing of multiple variable regions to generate high-quality results to a species level.

Introduction: Short-read amplicon sequencing studies have typically focused on 1-2 variable regions of the 16S rRNA gene. Species-level resolution is limited in these studies, as each variable region enables the characterisation of a different subsection of the microbiome. Although long-read sequencing techniques take advantage of all 9 variable regions by sequencing the entire 16S rRNA gene, they are substantially more expensive. This work assessed the feasibility of accurate species-level resolution and reproducibility using a relatively new sequencing kit and bioinformatics pipeline developed for short-read sequencing of multiple variable regions of the 16S rRNA gene. In addition, we evaluated the potential impact of different sample collection methods on our outcomes. Methods: Using xGen™ 16S Amplicon Panel v2 kits, sequencing of all 9 variable regions of the 16S rRNA gene was carried out on an Illumina MiSeq platform. Mock cells and mock DNA for 8 bacterial species were included as extraction and sequencing controls respectively. Within-run and between-run replicate samples, and pairs of stool and rectal swabs collected at 0-5 weeks from the same participants, were incorporated. Observed relative abundances of each species were compared to theoretical abundances provided by ZymoBIOMICS. Paired Wilcoxon rank sum tests and distance-based intraclass correlation coefficients were used to statistically compare alpha and beta diversity measures, respectively, for pairs of replicates and stool/rectal swab sample pairs. Results: Using multiple variable regions of the 16S ribosomal Ribonucleic Acid (rRNA) gene, we found that we could accurately identify taxa to a species level and obtain highly reproducible results at a species level. Yet, the microbial profiles of stool and rectal swab sample pairs differed substantially despite being collected concurrently from the same infants. Conclusion: This protocol provides an effective means for studying infant gut microbial samples at a species level. However, sample collection approaches need to be accounted for in any downstream analysis.

Co-designing a Health Journey Mapping resource for culturally safe health care with and for First Nations people.

Background Many healthcare professionals and services strive to improve cultural safety of care for Australia's First Nations people. However, they work within established systems and structures that do not reliably meet diverse health care needs nor reflect culturally safe paradigms. Journey mapping approaches can improve understanding of patient/client healthcare priorities and care delivery challenges from healthcare professionals' perspectives leading to improved responses that address discriminatory practices and institutional racism. This project aimed to review accessibility and usability of the existing Managing Two Worlds Together (MTWT) patient journey mapping tools and resources, and develop new Health Journey Mapping (HJM) tools and resources. Method Four repeated cycles of collaborative participatory action research were undertaken using repeated cycles of look and listen, think and discuss, take action together. A literature search and survey were conducted to review accessibility and usability of MTWT tools and resources. First Nations patients and families, and First Nations and non-First Nations researchers, hospital and university educators and healthcare professionals (end users), reviewed and tested HJM prototypes, shaping design, format and focus. Results The MTWT tool and resources have been used across multiple health care, research and education settings. However, many users experienced initial difficulty engaging with the tool and offered suggested improvements in design and usability. End user feedback on HJM prototypes identified the need for three distinct mapping tools for three different purposes: clinical care, detailed care planning and strategic mapping, to be accompanied by comprehensive resource materials, instructional guides, videos and case study examples. These were linked to continuous quality improvement and accreditation standards to enhance uptake in healthcare settings. Conclusion The new HJM tools and resources effectively map diverse journeys and assist recognition and application of strengths-based, holistic and culturally safe approaches to health care.

Extended interactive voice response telephony (IVR) for relapse prevention after smoking cessation using varenicline and IVR: a pilot study.

BACKGROUND: There is a significant resumption of smoking following smoking cessation using varenicline. Both smoking cessation medications and counseling have been shown to increase smoking quit rates at one year. Thus, the combination of varenicline and interactive voice response (IVR) telephony followed by extended IVR may further improve smoking cessation rates at one and two years. METHODS: 101 participants were recruited from the community via newspaper advertisement. They attended a group counseling session and were given smoking information booklets from the Canadian Cancer Society. After 12 weeks of varenicline and 9 IVR calls, all participants who had quit smoking were randomized into 2 groups matched by levels of motivation and addiction as per baseline questionnaire score. The intervention group continued to receive bi-weekly IVR support for weeks 13-52. The control group no longer received IVR. The primary end-point was self-reported abstinence and exhaled carbon monoxide levels of less than 10 ppm for weeks 12, 52 and 2 years. Data were analyzed by Fisher's exact test or Wilcoxon rank-sum test. RESULTS: Of the 101 participants, 44 (43%) had stopped smoking after 12 weeks of varenicline and 9 IVR calls. Of these, 23 (52%) were randomized to receive IVR calls from weeks 13 to 52.At 52 weeks, 26 (59%) participants remained smoke-free. Of the 23 with IVR, 12 (52.2%) stopped smoking compared to 14 of 21 (66.7%) without IVR. At 2 years, 40 of the 44 (90.9%) randomized participants were contacted and 24 of the 44 (54.5%) came in for testing. Fourteen (13% of the original cohort, 30% who were abstinent at 12 weeks and 53% who were abstinent at 52 weeks) remained smoke-free. Five of the 23 (21.7%) randomized to IVR and 9 of the 21 (42.9%) randomized to no IVR remained smoke-free at 2 years. CONCLUSIONS: In this pilot study of an apparently healthy population, extended IVR did not affect abstinence rates. There was no relapse prevention benefit in offering 9 months of continued IVR to subjects who had stopped smoking after receiving 3 months of varenicline and IVR treatment. TRIAL REGISTRATION: ClinicalTrial.gov: NCT00832806.

A review of the auditory-gut-brain axis.

Hearing loss places a substantial burden on medical resources across the world and impacts quality of life for those affected. Further, it can occur peripherally and/or centrally. With many possible causes of hearing loss, there is scope for investigating the underlying mechanisms involved. Various signaling pathways connecting gut microbes and the brain (the gut-brain axis) have been identified and well established in a variety of diseases and disorders. However, the role of these pathways in providing links to other parts of the body has not been explored in much depth. Therefore, the aim of this review is to explore potential underlying mechanisms that connect the auditory system to the gut-brain axis. Using select keywords in PubMed, and additional hand-searching in google scholar, relevant studies were identified. In this review we summarize the key players in the auditory-gut-brain axis under four subheadings: anatomical, extracellular, immune and dietary. Firstly, we identify important anatomical structures in the auditory-gut-brain axis, particularly highlighting a direct connection provided by the vagus nerve. Leading on from this we discuss several extracellular signaling pathways which might connect the ear, gut and brain. A link is established between inflammatory responses in the ear and gut microbiome-altering interventions, highlighting a contribution of the immune system. Finally, we discuss the contribution of diet to the auditory-gut-brain axis. Based on the reviewed literature, we propose numerous possible key players connecting the auditory system to the gut-brain axis. In the future, a more thorough investigation of these key players in animal models and human research may provide insight and assist in developing effective interventions for treating hearing loss.

Reporting of patient journey mapping in current literature: a scoping review protocol.

OBJECTIVE: This scoping review will assess the literature that documents or utilizes patient journey mapping methodologies in health care settings. It will also examine the reporting processes of studies that use this methodology. INTRODUCTION: Health care systems are complex and can be challenging for patients to navigate. Using patient journey mapping as a research method promotes a deeper understanding of patient experiences when navigating these systems. Patient journey mapping provides valuable insights into where systems are working well, where gaps in care exist, and how the system could respond to these gaps. INCLUSION CRITERIA: This review will consider peer-reviewed articles and publicly available academic literature documenting patient journey mapping methodologies. The review will also consider studies providing guidance and recommendations on how to report patient journey mapping studies in health care services and systems. METHODS: The proposed review will follow JBI guidance for scoping reviews. The following databases will be searched: MEDLINE, Embase, Emcare, PsycINFO, Scopus, Web of Science Core Collection, the Directory of Open Access Journals, Informit, and ProQuest Dissertations and Theses Global. The search will not be limited to year of publication but will be limited to studies reported in English. The PRISMA-ScR extension will be used to document the literature search. Two reviewers will screen titles, abstracts, and full-text articles. An extraction table will be used to extract relevant data from all included articles and to facilitate data analysis.

The biguanide polyamine analog verlindamycin promotes differentiation in neuroblastoma via induction of antizyme.

Deregulated polyamine biosynthesis is emerging as a common feature of neuroblastoma and drugs targeting this metabolic pathway such as DFMO are in clinical and preclinical development. The polyamine analog verlindamycin inhibits the polyamine biosynthesis pathway enzymes SMOX and PAOX, as well as the histone demethylase LSD1. Based on our previous research in acute myeloid leukemia (AML), we reasoned verlindamycin may also unblock neuroblastoma differentiation when combined with all-trans-retinoic acid (ATRA). Indeed, co-treatment with verlindamycin and ATRA strongly induced differentiation regardless of MYCN status, but in MYCN-expressing cells, protein levels were strongly diminished. This process was not transcriptionally regulated but was due to increased degradation of MYCN protein, at least in part via ubiquitin-independent, proteasome-dependent destruction. Here we report that verlindamycin effectively induces the expression of functional tumor suppressor-antizyme via ribosomal frameshifting. Consistent with previous results describing the function of antizyme, we found that verlindamycin treatment led to the selective targeting of ornithine decarboxylase (the rate-limiting enzyme for polyamine biosynthesis) as well as key oncoproteins, such as cyclin D and Aurora A kinase. Retinoid-based multimodal differentiation therapy is one of the few interventions that extends relapse-free survival in MYCN-associated high-risk neuroblastoma and these results point toward the potential use of verlindamycin in this regimen.

Interdependent nucleocytoplasmic trafficking and interactions of Dis3 with Rrp6, the core exosome and importin-alpha3.

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome-binding protein Dis3, a processive 3' to 5' exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N-terminal domain mutants of dDis3 abolish associations with the 'core' exosome, yet only reduce binding to the 'nuclear' exosome-associated factor dRrp6. We show that nuclear localization of dDis3 requires a C-terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co-precipitates the NLS-binding protein importin-alpha3. Surprisingly, dDis3 constructs that lack or mutate the C-terminal NLS retain importin-alpha3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin-alpha3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.

I'm Just a Person: Self-Labeling Following Sexual Assault.

OBJECTIVE: The purpose of this study was to examine college women's self-labeling as a victim or a survivor following a sexual assault and describe the relationship of self-labeling with mental health, self-blame, control over recovery, and help-seeking. METHODS: This cross-sectional study collected data in an online anonymous survey in November and December of 2018. Participants (N = 375) were recruited from two public universities, were 18- to 24-year-old undergraduate students, identified as female, and had experienced a sexual assault since entering college. RESULTS: Most respondents (46.4%, 174/375) chose labels other than victim or survivor. Statistically significant differences were found between choice of label (survivor, victim, or other) and depression, well-being, characterological self-blame, and perceived control over recovery. Short-answer responses revealed three major themes for alternative labels: choosing no label, normalizing, and seeking congruence. CONCLUSION: As when caring for a patient with any diagnosis, nurses and other healthcare providers should see a person-not a patient, a survivor, or a victim.

Innovative public health staff augmentation concepts during a global pandemic.

At the onset of the COVID-19 global pandemic, Florida's State Emergency Response Team's Emergency Support Function 8 (ESF-8) Health and Medical Staffing Unit faced a surge of personnel requests from the field. The unit found that, given the scope of requests, standard disaster staffing practices could not always accommodate the requirements of the requests. With full support of leadership, the ESF-8 Staffing Unit developed new and innovative practices to streamline the cumbersome hiring process including coordinating with internal and external partners to expedite staff identification and implementing just-in-time training.